ABSTRACT Patients with Down syndrome (DS, trisomy 21, T21) demonstrate a spectrum of clinical phenotypes due to an extra copy of chromosome 21 (HSA21). The clinical spectrum encompasses intellectual disability, early onset Alzheimer?s disease, congenital heart defects, and blood abnormalities including childhood leukemia. DS phenotypes are likely related to alteration of gene expression due to the extra copy of HSA21, and understanding the genomic determinants that contribute to the different phenotypes is a major objective in DS research. Our efforts will address several existing challenges that include: i. murine models do not recapitulate all of the human DS phenotypes, ii. manipulation of HSA21 gene dosage in murine and human DS cellular models have relied on methods that alter expression in non-physiologic doses, and iii. transcriptomic studies between normal and T21 tissues are challenged by considerable ?noise? from inherent gene expression variation among individuals. In this application, we will address these challenges by using innovative technologies to create, genetically manipulate, and analyze patient-derived induced pluripotent stem cells (iPSCs). We will use a defined set of T21 iPSC lines and CRISPR-CAS9 gene editing to produce isogenic cell lines that differ only by copy number of HSA21 or specific candidate genes. Using isogenic T21 and euploid iPSCs, we will test whether T21 disrupts overall chromosomal architecture that results in genome-wide gene expression dysregulation. We will perform studies in gene expression, chromosome architecture, DNA methylation, and chromatin signatures to provide a comparative view of genome-wide transcriptome and chromatin contacts in isogenic T21 and euploid cells. Further studies will examine whether abnormalities are due to the overexpression of one or more specific HSA21 genes, or as a consequence of heterozygous mutations in CTCF or cohesion components identified in DS patients with leukemia. Our goal is to produce novel, medically relevant knowledge that advances our understanding of gene dosage imbalance in DS, with a particular interest in DS-associated leukemia and potential new treatments. We aim to not only provide insight into DS abnormalities, but may have broader implications for other diseases associated with aneuploidy.